To investigate how actin and microtubules function in vivo, we are using the Natural Killer (NK) lymphoid cell as a model cell system. This system offers physiological relevance to human biology and disease, ease of use, and a multiplicity of cytoskeleton-based functions downstream of diverse signaling pathways. In previous studies, we discovered that the cortactin homologue HS1 is essential for many actin-based functions and signaling pathways. We found that Arp2/3 is necessary for assembly of the actin-based lytic synapse and that the formin Dia1 appears to capture microtubule plus ends at the lytic synapse, which is required for the polarized secretion of lytic granules onto target cells. Aim 1. Functions of the Multi-domain Cortactin Homologue HS1. a. Distinct functions of two Src-phosphorylated tyrosines. We discovered that Tyr 378 is necessary for NK cell chemotaxis, while Tyr 397 is necessary for target cell killing. We will search for specific ligands of these individual phosphotyrosines, investigate the functional roles of the ligands and test the significance of their interactions with HS1. b. Functions of HS1 domains. HS1 has multiple domains with different interaction partners. We will study the functions of these domains and the physiological relevance of their molecular interactions. Aim 2. Microtubule Plus-end Capture, MTOC Polarization and Lytic Granule Movement. a. Microtubule capture by the lytic synapse. The formin Dia1 appears to be necessary for the capture of dynamic microtubule plus ends at the lytic synapse, acting in concert with APC and EB1. We will investigate the molecular mechanisms that underlie microtubule capture, using a combination of molecular genetics and cell biology approaches. b. Positioning of the MTOC. We hypothesize that the MTOC moves to a forward position in association with assembly of the lytic synapse. We will determine whether microtubule capture or other mechanisms are involved in positioning the MTOC. c. Lytic granule movement. Lytic granules line up along microtubules in the NK cell, and those microtubules are directed toward the target cell. In contrast to an influential previous study, we hypothesize that plus-end directed transport of lytic granules will be necessary for their secretion onto the target cell. We will investigate the molecular mechanism of plus-end transport, testing various kinesins. d. Role of actin in granule transport and fusion. We hypothesize that actin filaments may play positive or negative roles in how lytic granules are transported to the lytic synapse and then fuse with the plasma membrane. We will investigate potential roles by observing transport and manipulating the actin cytoskeleton. PUBLIC HEALTH RELEVANCE: This research will help us understand the molecular mechanisms by which certain lymphocytes, called Natural Killer cells, are able to find and kill cancer cells and virus-infected cells. This fundamental information may serve as the basis for the development of new therapies for human diseases involving the molecules and processes that are discovered and illuminated.